ANALISIS IN SILICO PERANCANGAN crRNA CRISPR DENGAN TARGET SEKUENS PENYANDI SQUALENE SYNTHASE PADA Artemisia annua Linn.

Ivan Tjahja Pranata (1)


(1) 


Abstract


Artemisinin adalah salah satu jenis fitofarmaka yang penting oleh karena beberapa potensi medisnya. Meskipun demikian, produktivitas Artemisia annua di Indonesia secara umum masih berkisar di bawah 0,5% sehingga secara komersial belum layak untuk dibudidayakan. Salah satu faktor utama yang menghambat produktivitas artemisinin adalah keberadaan gen SQS yang berkompetisi dengan gen ADS dalam penggunaan susbtrat farnesil difosfat. Kompetisi ini menurunkan kadar prekursor kunci bagi pembentukan senyawa artemisinin. Teknik penyuntingan genom CRISPR berbasis non-GMO dapat diaplikasikan untuk mengatasi hambatan ini. Mekanisme knockout yang disebabkan oleh sistem CRISPR pada gen SQS dapat menekan tingkat kompetisi substrat yang terjadi pada jalur biosintesis artemisinin sehingga diharapkan meningkatkan kadar artemisinin. Sebelum dilakukan transformasi secara in vitro, perlu dilakukan analisis in silico untuk pembuatan model crRNA sintetis. Dalam penelitian ini, digunakan 2 jenis perangkat lunak berbasis bioinformatika yaitu E-CRISP dan CRISPRdirect untuk pelacakan on-target dan off-target. Berdasarkan hasil perancangan yang telah dilakukan, 10 desain crRNA sintetis berhasil dibentuk dengan model crRNA_F5 sebagai desain yang paling optimal. Model crRNA_F5 ini memiliki sekuens protospacer 5’- GGTGTAGTCAAATTGAGACG-3’ dan sekuens PAM 5’-TGG-3’. Pemilihan desain crRNA_F5 dipertimbangkan dari nilai efficacy sebesar 78.57, skor Doench dan skor Xu tertinggi, serta minimalisasi jumlah off-target pada sekuens 8mer pada jumlah 259. Model ini sama sekali tidak memiliki off-target pada sekuens 20mer dan 12mer.


Keywords


crRNA, SQS, E-CRISP, CRISPRdirect, knockout

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